The long-term objective of this application is to develop methods for the efficient determination of atomic- resolution three-dimensional (3D) structures of large biological complexes in their native, non-crystalline states. The emerging technology of cryo-electron microscopy and 3D reconstruction (collectively cryoEM) offers great promise for such structural studies. In the current funding period, the PI's group has developed data processing methods, implemented software, and validated these advances by determination of cryoEM structures of a number of large complexes to near-atomic resolution. We hypothesize that improvements in cryoEM technique, coupled with powerful computational tools, can be developed to create and process terabytes of image data for determination of atomic models of large complexes. Whereas we aimed and succeeded in the current funding period to obtain cryoEM structures at near-atomic resolution, the overall goal of this renewal application is to obtain structures at atomic resolution. In the renewal, we propose to improve both imaging itself and computation to bring resolution to 2.5E, permitting identification of amino acids and ultimately atomic resolution. In the renewal period, we therefore focus on improvement of imaging and methods for image acquisition and correction (Aim #1), making cryoEM structure determination hundreds of times more efficient and affordable (Aim #2), extending our success with icosahedral objects to helical ones (Aim #3), improvement of cryoEM-specific atomic-model refinement software that takes advantage of phase as well as amplitude data and that can handle the vastly greater amounts of data required for atomic resolution 3D reconstruction (Aim #4), and validation by applying these new methods for atomic structure determination to a small icosahedral hepatitis B virus (HBV) core, a large icosahedral aquareovirus and the helical tobacco mosaic virus (TMV) (Aim #5). Our overriding principle in achievement of all of these aims is that the optimized methods we create should permit them to be used routinely, reproducibly, and affordably. A successful outcome of this renewal project will remove the final obstacles towards determination of atomic- resolution structures for large complexes by cryoEM and will have great impact on many areas of biomedical research. This achievement will complement other structural methods, particularly X-ray crystallography of purified proteins that are amenable to crystallization and NMR of small molecules in solution. Specifically, this achievement will enable investigators to place individual proteins within the structural context of the larger assembly and to visualize native shapes and physical chemical interactions among the parts of the complex. Moreover, the ability to look at large complexes at atomic resolution will permit visualization of complexes bound to antibodies, receptors, and drugs.